Biomolecules can be labeled with any of a variety of reagents, including radionuclides, toxins, vitamins, fluorescent compounds and chelating agents. A labeling reagent may be incorporated as a constituent of a biomolecule, for example by metabolic labeling or by nick translation, or may be attached to a biomolecule by a covalent bond or another intermolecular force. Examples of the latter category of labeling method include the use of isothiocyanate derivatives of fluorochromes to render antibodies fluorescent, the use of photoactive derivatives of biotin to label nucleic acids and the use of oxidative or enzyme-mediated reactions to attach iodine onto proteins at tyrosine residues. The labeling procedure can be as simple as mixing a biomolecule and a labeling reagent.
U.S. Pat. No. 4,707,352 discloses a labeling method which comprises the step of contacting an unlabeled compound, consisting of a chelating agent conjugated to a biomolecule, with an ion transfer material to which is bound a radiometal. The affinity of said ion transfer material for the radiometal is less than the affinity of said chelating agent for said metal. In the example a column containing an ion exchange resin loaded with .sup.63 Ni is used. A chelator conjugate is passed through the column and eluted as the radiolabeled chelator conjugate. The components to perform the labeling method may be incorporated into kits.
At the conclusion of a labeling reaction, it is often desirable to purify the labeled biomolecule, the product, by separating any reactant, particularly unreacted labeling reagent, from said product. Presence of unbound labeling reagent can confound outcomes by associating with irrelevant molecules (non-specific binding) or by contributing to the background.
Because many labeling reagents are small molecules or elements, common methods for separating product from unbound reagent rely on size or weight differential. Thus, size exclusion chromatography or dialysis may be used. If there is a charge difference between the product and reactant, ion exchange chromatography is a suitable separation method.
A method for purifying radiolabeled antibody combining ion exchange and size exclusion resins is disclosed in U.S. Pat. No. 4,454,106. A 9 cm column is made comprising 1 ml of an ion retarding resin above 1 ml of a 200-400 mesh cation exchange resin above 7 ml of gel filtration medium capable of fractionating particles 1,500-25,000 daltons in weight. The column is equilibrated with a buffer consisting of 200 mM sodium chloride and 10 mM MES at pH 6.0. In the related U.S. Pat. No. 4,472,509, the preferred bed for purifying technetium-labeled antibodies is the three component bed described above modified to include 1 ml of an anion exchange resin situated below the cation exchange resin and above the gel filtration medium.
Mukkala et al. (Anal Biochem (1989) 176:319-325) labeled IgG with Eu.sup.3+ using bridging chelators. The labeled antibody product was purified from the reactants by passing the reaction mixture over a combined 1.5.times.5 cm Sephadex G-50 (Pharmacia Fine Chemicals; dextran beads) column and a 1.5.times.30 cm Trisacryl GF2000 (Reactifs IBF; polyacrylamide beads) column.
Esteban et al. (J. Nucl Med (1987) 28:861-870) compared four protocols for purifiying .sup.111 In-labeled antibody at completion of the labeling procedure. They divided a single labeling preparation into four equal portions. One aliquot was treated with excess EDTA in solution without subsequent separation. Another aliquot was passed over a 1.times.8 cm gel exclusion (Sephadex G-50 fine) column. The third aliquot was injected onto a 7.5 mm.times.30 cm HPLC (TSK 3000) column and the final aliquot was treated sequentially over the G50 column and then the TSK 3000 column. The poorest results were obtained with the EDTA treatment, the G-50 column was marginally better, the HPLC-purified labeled antibody had a tumor:liver ratio three times that of the EDTA-purified aliquot and the best results were obtained with the G-50/TSK 3000 combination. The authors concluded that the widely used EDTA method was inefficient in producing clean preparations and other purification methods should be considered if one wants to minimize background.
U.S. Pat. No. 4,775,638 discloses a single vial technique for radiolabeling antibody. The method comprises introducing radioisotope into a sealed vessel in which the inner surface of said vessel is coated with a catalyst; introducing antibody into said vessel; incubating the mixture; introducing into said vessel an ion exchange resin which absorbs radioisotope not bound to antibody; withdrawing the mixture; and separating the resin from the supernatant. The preferred resin is an anion exchanger such as AG 1-X8 (Bio-Rad). Although the method is directed primarily to radio-iodination procedures, the inventor surmised that the catalyst-mediated attachment of radioisotope to antibody and the subsequent purification of said labeled antibody might be adapted for .sup.67 Ga and .sup.111 In labeling by chelation.
Notwithstanding the variety of separation methods available to the artisan, a systematic limitation constrains the use of labeled biomolecules in procedures demanding high sensitivity. That limitation is the sometimes low efficacy of removing unbound labeling reagent of many current methods in the art. A clear example is the use of radiolabeled anti-cancer antibodies in situ for the detection of malignant growths, a method known as radioimmunoscintigraphy. For various reasons not related directly to the instant invention, often, only a small amount of antibody binds to a malignancy. Thus, the signal is difficult to discern even under ideal conditions. It is not uncommon for diagnosis to be rendered impossible because of high background. Accordingly, one way to assure or enhance detection is using labeled antibody that is significantly free of unbound radionuclides.
A further limitation to the use of gel exclusion chromatography is the propensity of IgM antibodies to bind nonspecifically and at times irreversibly to the column matrix. See for example Halpern et al., J. Nucl Med (1988) 29:1688-1696.
Another limitation relates to the prolonged time frame of most procedures. Many of the radionuclides and particularly the radiometals have a short half-life, which if not days, often is a matter of hours. Thus rapid purification enhances the specific activity of a labeled biomolecule preparation.
Furthermore, the separation procedures recited above are not without additional shortcomings. Many require costly equipment and skilled technicians, are prone to biologic contamination, require close monitoring, are not amenable to scale-up and the like. The equipment may be difficult and costly to decontaminate in the event of radioactive spills.
Resolution of the above-noted problems provided the motivation for the instant invention. Disclosed herein is a means for obtaining a higher degree of separation of labeled product from unbound reactant than that achieved using current procedures. The instant method is advantageous for several reasons, including simplicity and inexpensiveness. Additionally the method is not prone to biologic contamination, does not affect the bioactivity of the biomolecule, offers high concentrated yields, can be used in a standard hospital laboratory by nursing staff, is easy to dispose of after use, has a long shelf life and is adaptable for use with a variety of labeling reagents and biomolecules.